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United States Patent |
5,714,164
|
Renimel
,   et al.
|
February 3, 1998
|
Method for treatment of allergic disorders and cosmetic compositions
using cucurbitine
Abstract
The invention relates to the use of cucurbitine or extracts of
Cucurbitaceae pips for the preparation of a cosmetic or pharmaceutical, in
particular dermatological, composition having antiallergic activity, or
for the preparation of cosmetic or pharmaceutical compositions having a
reduced risk of being allergenic.
Inventors:
|
Renimel; Isabelle (Trainou, FR);
Andre; Patrice (Neuvilles Aux Bois, FR)
|
Assignee:
|
Parfums Christian Dior (Paris, FR)
|
Appl. No.:
|
486621 |
Filed:
|
June 7, 1995 |
Foreign Application Priority Data
Current U.S. Class: |
424/450; 424/43; 424/45; 424/46; 424/401; 424/427; 514/359; 514/826; 514/861 |
Intern'l Class: |
A61K 009/127; A61K 031/40 |
Field of Search: |
424/450,401,427,43,45,46
514/359,826,861
|
References Cited
U.S. Patent Documents
4255418 | Mar., 1981 | Bailey | 424/145.
|
4421746 | Dec., 1983 | Kojima | 424/195.
|
4511559 | Apr., 1985 | Szendrei | 514/54.
|
4883659 | Nov., 1989 | Goodman | 424/78.
|
4942153 | Jul., 1990 | Fernandez | 514/2.
|
4980038 | Dec., 1990 | Watanabe | 204/157.
|
5039516 | Aug., 1991 | Goodman | 424/59.
|
5118507 | Jun., 1992 | Clement | 424/401.
|
5128139 | Jul., 1992 | Byron | 424/450.
|
5164182 | Nov., 1992 | Meybeck | 424/195.
|
5165935 | Nov., 1992 | Andre | 424/450.
|
Foreign Patent Documents |
2522500 | Sep., 1983 | FR.
| |
Other References
World Patents Index Latest, AN 87-329987 ›47!, Derwent Publications Ltd.,
London, GB, & JP, S, 62234013 (Osaka Yakuhin Kenky), 14 Oct. 1987.
Chemical Abstracts, vol. 82, No. 23, 1975, p. 40, abstract 149446a,
Pharmacological (anthelminthic) study of Cucurbita . . . , A.E. Gonzalez
et al.
Scienta Sinica, vol. X, No. 7, 1961, T.-T. Sun et al, "Chemical studies on
cucurbita moschata duch", pp. 852-859.
Journal of the Chemical Society, Chemical Communications, 1973, H.J.
Monteiro, "New synthesis of the Amino-acid (+)-Cucurbitine", p. 2.
Chemical and Pharmaceutical Bulletin, vol. 35, No. 9, Sep. 1987, Y.
Morimoto et al, "Enzymes and catalysts. II. Pig liver . . . ", pp.
3845-3849.
J. Falbe, "Methoden der Organischen Chemie", vol. E5, Georg Thieme Verlag
pp. 534-543, 1981.
World Patents Index Latest, 1977, AN 77-39162& ›22!, Derwent Publications
Ltd. 52051033 (Ogawa) 23 Apr. 1977.
World Patents Index Latest, 1987, AN 87-273386 ›39!, Derwent Publications
Ltd. 62087241 (Lion Corp.) 21 Apr. 1987.
World Patents Index Latest, 1987, AN 87-105806 ›15!, Derwent Publications
Ltd. 52051033 (Shiseido) 9 Mar. 1987.
World Patents Index Latest, 1987, AN 87-032891 ›05!, Derwent Publications
Ltd. 62087241 (Shiseido) 19 Dec. 1986.
Price Pathophysiology, Clinical Concepts of Disease Processes, 1986, pp.
36-37.
Webster's New World Dictionary, 1988, p.36.
|
Primary Examiner: Kishore; Gollamudi S.
Attorney, Agent or Firm: Sherman and Shalloway
Parent Case Text
This application is a continuation of application Ser. No. 08/108,601,
filed Sep. 28, 1993, now abn. which is a 371 of PCT/Fr92/00164 filed Feb.
24, 1992.
Claims
We claim:
1. A method for treating histamine dependent allergic manifestations in a
human being or an animal in need thereof, which comprises administering to
the human being or animal a composition containing a histamine dependent
allergy treating agent which consists essentially of an histamine
inhibiting effective amount of Cucurbitine.
2. The method of claim 1, wherein Cucurbitine is selected from the group
consisting of a pharmaceutically or cosmetically acceptable salt or ester
of Cucurbitine.
3. The method of claim 1, wherein Cucurbitine is present in laevorotatory
form.
4. The method of claim 1, comprising administering said composition
containing from 0.001% to 10% by weight of Cucurbitine based on the total
composition.
5. The method of claim 1, comprising administering said composition
containing from 0.01% to 10% by weight of Cucurbitine based on the total
composition.
6. The method of claim 1, comprising administering said composition
containing from 0.01% to 5% by weight of Cucurbitine based on the total
composition.
7. The method of claim 1, comprising administering Cucurbitine at least in
part encapsulated in liposome vesicles.
8. The method of claim 1, wherein said Cucurbitine is selected from the
group consisting of (-)Cucurbitine, Cucurbitine mono hydrobromide,
Cucurbitine dihydrobromide, Cucurbitine mono hydrochloride, Cucurbitine
dihydrochloride, Cucurbitine methyl ester and Cucurbitine ethyl ester.
9. The method of claim 1, comprising administering said composition
containing Cucurbitine as an aerosol.
10. A method for decreasing the histamine dependent allergenic potential of
a cosmetic or pharmaceutical composition, comprising incorporating in said
composition an effective amount of Cucurbitine in at least substantially
pure form, so that the final composition displays a reduced risk of being
allergenic.
11. The method of claim 10, wherein Cucurbitine is selected from the group
consisting of pharmaceutically or cosmetically acceptable salt or ester of
Cucurbitine.
12. The method of claim 10, wherein Cucurbitine is present in laevorotatory
form.
13. The method of claim 10, comprising incorporating Cucurbitine in said
composition in an amount of from 0.001% to 10% by weight of the total
composition.
14. The method of claim 10, comprising incorporating Cucurbitine in said
composition in an amount of from 0.01% to 10% by weight of the total
composition.
15. The method of claim 10, comprising incorporating Cucurbitine in said
composition in an amount of from about 0.01% to 5% by weight of the total
composition.
16. The method of claim 10, comprising administering Cucurbitine at least
in part encapsulated in liposome vesicles.
17. The method of claim 10, wherein said Cucurbitine is selected from the
groups consisting of (-)Cucurbitine, Cucurbitine mono hydrobromide,
Cucurbitine dihydrobromide, Cucurbitine mono hydrochloride, Cucurbitine
dihydrochloride, Cucurbitine methyl ester and Cucurbitine ethyl ester.
18. A method for decreasing histamine concentration in the blood serum and
tissues of a person in need thereof comprising systemically administering
to said person from about 0.1 to 20 mg/kg/day of at least substantially
pure Cucurbitine.
19. The method of claim 18, wherein Cucurbitine is selected from the group
consisting of a pharmaceutically acceptable salt or ester of Cucurbitine.
20. The method of claim 18, wherein Cucurbitine is present in laevorotatory
form.
21. The method of claim 18, comprising systemically administering
Cucurbitine in a composition comprising from 0.001% to 10% by weight of
said at least substantially pure Cucurbitine based on the total
composition.
22. The method of claim 18, comprising systemically administering
Cucurbitine in a composition comprising from 0.01% to 5% by weight of said
at least substantially pure Cucurbitine based on the total composition.
23. The method of claim 18, comprising systemically administering
Cucurbitine in a composition comprising from 0.01% to 5% by weight of said
at least substantially pure Cucurbitine based on the total composition.
24. The method of claim 18, comprising systemically administering
Cucurbitine at least in part encapsulated in liposome vesicles.
25. The method of claim 19, wherein said Cucurbitine is selected from the
group consisting of (-)Cucurbitine, Cucurbitine mono hydrobromide,
Cucurbitine dihydrobromide, Cucurbitine mono hydrochloride, Cucurbitine
dihydrochloride, Cucurbitine methyl ester and Cucurbitine ethyl ester.
26. The method of claim 18, comprising systemically administering
Cucurbitine as an aerosol.
27. A method of treating histamine dependent respiratory allergic disorders
of a human being or an animal comprising administering to said human being
or animal at least substantially pure Cucurbitine in an amount which is
effective for treating said respiratory allergic disorders.
28. The method of claim 27, wherein said respiratory allergic disorders are
selected from the group consisting of bronchial asthma and asthmoid
bronchitis.
29. The method of claim 27, wherein Cucurbitine is selected from the group
consisting of a pharmaceutically acceptable salt or ester of Cucurbitine.
30. The method of claim 27, wherein Cucurbitine is present in laevorotatory
form.
31. The method of claim 29, comprising administering Cucurbitine in a
composition comprising from 0.001% to 10% by weight of said at least
substantially pure Cucurbitine based on the total composition.
32. The method of claim 27, comprising administering Cucurbitine in a
composition comprising from 0.01% to 10% by weight of said at least
substantially pure Cucurbitine based on the total composition.
33. The method of claim 27, comprising administering Cucurbitine in a
composition comprising from 0.01% to 5% by weight of said at least
substantially pure Cucurbitine based on the total composition.
34. The method of claim 27, comprising administering Cucurbitine at least
in part encapsulated in liposome vesicles.
35. The method of claim 27, wherein said cucurbitine is selected from the
group consisting of (-)Cucurbitine, Cucurbitine mono hydrobromide,
Cucurbitine dihydrobromide, Cucurbitine mono hydrochloride, Cucurbitine
dihydrochloride, Cucurbitine methyl ester and Cucurbitine ethyl ester.
36. The method of claim 27, comprising administering Cucurbitine as an
aerosol.
37. A method for the cosmetic treatment of skin with a histamine dependent
allergic tendency, comprising applying to said skin a cosmetic composition
containing at least substantially pure Cucurbitine.
38. The method of claim 37, wherein Cucurbitine is selected from the group
consisting of a cosmetically acceptable salt or ester of Cucurbitine.
39. The method of claim 37, wherein Cucurbitine is present in laevorotatory
form.
40. The method of claim 37, comprising administering Cucurbitine in a
composition comprising from 0.001% to 10% by weight of said at least
substantially pure Cucurbitine based on the total composition.
41. The method of claim 37, comprising administering Cucurbitine in a
composition comprising from 0.01% to 10% by weight of said at least
substantially pure Cucurbitine based on the total composition.
42. The method of claim 37, comprising administering Cucurbitine in a
composition comprising from 0.01% to 5% by weight of said at least
substantially pure Cucurbitine based on the total composition.
43. The method of claim 37, comprising administering Cucurbitine at least
in part encapsulated in liposome vesicles.
44. The method of claim 37, wherein said Cucurbitine is selected from the
group consisting of (-)Cucurbitine, Cucurbitine mono hydrobromide,
Cucurbitine dihydrobromide, Cucurbitine mono hydrochloride, Cucurbitine
dihydrochloride, Cucurbitine methyl ester and Cucurbitine ethyl ester.
45. The method of claim 37, wherein said skin is a dry skin.
46. The method of claim 37, wherein said skin is a sensitive skin.
47. The method of claim 1, wherein said treatment is selected from the
group consisting of bronchial asthma, hayfever, spasmodic tracheitis,
rhinitis, urticaria, eczema, red blotches, pruritus, Quincke's oedema, and
allergic conjunctivitis.
Description
The present invention relates essentially to the use of cucurbitine for the
preparation of antiallergic cosmetic or pharmaceutical, in particular
dermatological, compositions, and to a process involving application
thereof.
Cucurbitine, or 3-amino-3-pyrrolidinecarboxylic acid, of the following
formula I:
##STR1##
is a natural, water-soluble amino acid found in Cucurbitaceae (see V. H.
Mihranian et al., LLOYDIA (1968), 31 (1) 23-29).
Cucurbitine is known as an antiparasitic, especially as an anthelmintic
against Schistosoma japonicum. (Morimoto Y. et al., Chem. Pharm. Bull.
(1987) 35 (9) 3845-3849).
Cucurbitine may be obtained by means of extraction in laevorotatory form,
or synthetically in racemic form. Among the various methods of synthesis
of cucurbitine, special mention may be made of the synthesis method of H.
J. Monteiro, J. Chem. Soc., Chem. Commun. (1973) 2. This method leads to
only relatively low yields of racemic cucurbitine. Another synthesis
method enables the two optical isomers of cucurbitine to be obtained
separately. This is the method of Morimoto et al., Chem. Pharm. Bull.
(1987) 35 (9) 3845-3849, which is a stereospecific enzymatic method of
synthesis by the use of a pig liver esterase. This method is, however,
complicated, and necessitates a relatively large number of steps.
It has now been discovered, unexpectedly, that cucurbitine inhibits the
formation of histamine, a well-known mediator of allergies, and hence
displays valuable hypohistaminaemic activity. This hypohistaminaemic
activity results from the inhibitory action of cucurbitine on histidine
decarboxylase, which is the enzyme responsible for the conversion of
histidine to histamine. As a result, the administration of cucurbitine
contributes to decreasing the histamine concentration in the blood serum
and tissues.
This constitutes a considerable technical advance, since allergic
manifestations, in particular pulmonary and cutaneous allergies, are
nowadays causing many problems for therapists, who have a limited number
of active substances at their disposal and, in addition, some of these
substances can display side-effects. Thus, a considerable need
consequently exists for the development of a new preventive and curative
composition for allergies.
Thus, the main objective of the present invention is to solve the technical
problem that consists in providing an approach enabling the formation of
histamine, a mediating agent in the context of allergic manifestations, to
be inhibited in order to enable allergic manifestations to be prevented
and treated.
According to another aspect, the main objective of the present invention is
to solve the technical problem that consists in providing an approach
enabling allergic manifestations to be prevented and treated.
The object of the present invention is also to solve the new technical
problem that consists in providing an approach enabling the allergenic
potential of cosmetic or pharmaceutical, in particular dermatological,
compositions to be decreased.
The object of the present invention is also to solve the new technical
problem that consists in providing an approach enabling the synthesis of
cucurbitine to be carried out by a simple synthesis process, necessitating
a minimum number of steps, in good yields.
The present invention enables all these technical problems to be solved
simply, reliably and reproducibly of a cosmetic or pharmaceutical, in
particular dermatological, composition having antiallergic activity.
The invention will now be described in greater detail with reference to the
following description and preferred embodiments and with the assistance of
the accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a Michaelis curve of the inhibitory activity with respect to
enzyme histidine decarboxylase (HDC) for each of control, tritoqualine and
cucurbitine in terms of initial rate versus amount histidine; and
FIG. 2 is a graphic plot of an RIA assay for antihistaminic activity of
cucurbitine, according to this invention, in comparison to that of
tritoqualine and a control.
According to a particular variant of embodiment, the preparation in
question is of a cosmetic or pharmaceutical, in particular dermatological,
composition intended for the prevention or symptomatic treatment of
allergic manifestations, irrespective of their origin and their point of
application, in particular the bronchi, skin and eye. Thus, the said
composition is intended, in particular, for the prevention or symptomatic
treatment of allergic or exercise-induced bronchial asthma, hayfever,
spasmodic tracheitis and rhinitis, urticaria, other allergic eruptions,
eczema, red blotches or skin irritations of allergic origin, pruritus,
Quincke's oedema, allergic conjunctivitis and also allergic reactions of
medicinal origin.
In the more particular field of cosmetology, the said composition is
intended advantageously for lines of products which are hypoallergenic or
for sensitive or irritable skins.
Cucurbitine may be used either in free form, or in the form of one of its
cosmetically or pharmaceutically, in particular dermatologically,
acceptable salts or esters. The abovementioned salts and esters may be
prepared by conventional processes which are well known to a person
skilled in the art. Among salts, the mono- and dihydrobromide and the
mono- and dihydrochloride may be mentioned. Among esters, the methyl ester
and the ethyl ester may be mentioned.
According to an advantageous variant, the abovementioned plant extract
containing cucurbitine is an extract of Cucurbitaceae, especially of
Cucurbita maxima Duch., of Cucurbita pepo L. or of Cucurbita moschata
Duch; preferably, it is an extract of pips or of fruit pulp. As a further
preference, it is an extract of Cucurbitaceae pips.
According to a particular variant, the abovementioned plant extract is an
extract of Cucurbitaceae fruit pulp containing at least 0.5% by weight of
cucurbitine.
According to an advantageous variant of embodiment, cucurbitine or one of
its cosmetically or pharmaceutically, in particular dermatologically,
acceptable salts or esters is present at a concentration of 0.001% to 10%,
and preferably 0.01 to 5%, by weight of the total composition.
According to a second aspect, the present invention also relates to a
cosmetic composition, characterized in that it comprises, as active
ingredient, cucurbitine or one of its cosmetically acceptable salts or
esters, or a plant extract containing it, where appropriate in a
cosmetically acceptable excipient, vehicle or carrier.
According to an advantageous variant, the abovementioned plant extract is
an extract of Cucurbitaceae as defined above.
According to a preferred variant of embodiment, cucurbitine or one of its
salts or esters is present in an amount which is effective for displaying
antiallergic activity, especially at a concentration of 0.001% to 10%, and
preferably 0.01% to 5%, by weight of the total composition.
According to a third aspect, the present invention relates to a
pharmaceutical, in particular dermatological, composition preferably
having antiallergic activity, characterized in that it contains
cucurbitine or one of its pharmaceutically acceptable salts or esters in
an amount which is effective for displaying antiallergic activity,
especially for preventing or treating allergic manifestations, in
particular in the bronchi, skin and eye, where appropriate in a
pharmaceutically acceptable excipient, vehicle or carrier.
According to a variant, the abovementioned cosmetic and pharmaceutical
compositions contain an extract of plant origin as defined above.
According to a fourth aspect, the present invention further relates to a
process for decreasing the allergenic potential of a cosmetic or
pharmaceutical, in particular dermatological, composition, characterized
in that an effective amount of cucurbitine, in free form or in the form of
one of its cosmetically or pharmaceutically acceptable salts or esters, or
of a plant extract containing it as defined above, is incorporated in the
said composition so that the final composition presents a reduced risk of
being allergenic.
In the context of any one of the above aspects, the preferred weight
concentration of cucurbitine, or of its salts or esters, for compositions
for topical use, is between 0.001% and 10%, and as a further preference
between 0.01% and 5%.
For compositions intended for administration systemically (such as orally,
parenterally, rectally, by inhalation, etc.), the cucurbitine
concentration is not critical and can reach, for example, 60% of the
composition. The dosage in man will generally be between 0.1 mg/kg/day and
20 mg/kg/day, and preferably between 1 mg/kg/day and 15 mg/kg/day.
Moreover, natural cucurbitine, of laevorotatory form, or its salts or
esters, is/are generally used. Especially advantageous sources of
laevorotatory cucurbitine are the pulp and pips of Cucurbitaceae,
especially of the species Cucurbita pepo L., Cucurbita maxima Duch. and
Cucurbita moschata Duch. However, the racemic form of cucurbitine or of
its salts or esters may also be used.
Moreover, cucurbitine may be used in pure form, or in the form of extracts,
according to any one of the extraction procedures known to a person
skilled in the art. Especially advantageous extraction procedures are
described in the publication of Valentine H. MIHRANIAN et al. in lloydia
1968, 31, (1) 23-29, which is incorporated herein by reference, especially
page 24. This process advantageously provides for treatment of
decorticated and defatted Cucurbitaceae seeds with water, which is
advantageously heated to at least 50.degree. C. with constant stirring for
several hours. Thereafter the mixture is centrifuged, the supernatant is
collected and the residues are re-extracted one or more times in a similar
manner with several portions of heated water. The supernatant and washings
are combined and then treated by adding to an equivalent volume of 85%
ethanol to precipitate proteinaceous matter in suspension, and the whole
is kept in a refrigerator overnight. The mixture is then centrifuged and
the supernatant is collected. The alcohol may be removed by distillation,
for example in a rotary evaporator. The aqueous solution may be used as it
is, or alternatively passed thereafter through a chromatography column for
example of the Dowex 50W-X-8 type, for example measuring 75.times.2.2.cm.
The column is washed with approximately 200 ml of water, and then eluted
with 1% aqueous ammonium hydroxide solution until the effluent gives a
negative response to the ninhydrin test. The eluate may be evaporated to
dryness under reduced pressure, heating where appropriate. The syrupy
residue may, again, be used as it is, or alternatively treated again with
hot water and advantageously added to an at least equivalent volume of 95%
ethanol. Thereafter, the mixture may be acidified dropwise with an acid to
bring the pH of the solution to a pH approximately equal to 5, for example
with 60% perchloric acid. It may then be placed in a refrigerator for
several days to obtain a precipitate of cucurbitine perchlorate. This
precipitate can then be dissolved in a few milliliters of water and, where
appropriate, passed through a chromatography column, preferably of the
Amberlite CG-45 type, for example measuring 20.times.1.5 cm. Evaporation
of the eluate under reduced pressure gives cucurbitine in the
substantially pure state.
Thus, the above process makes it possible to obtain either cucurbitine in
the pure state, or extracts having variable cucurbitine contents. In the
case where cucurbitine is used in the form of an extract of Cucurbitaceae,
the cucurbitine content is preferably equal to at least 0.5% by weight of
the extract. A preferred source for obtaining cucurbitine consists of the
pips of the Cucurbita species such as Cucurbita pepo L., Cucurbita maxima
Duch. and Cucurbita moschata Duch.
According to a fifth aspect, the present invention also covers a process
for the treatment of a human being or an animal for preventing or treating
allergic manifestations, characterized in that an amount of cucurbitine or
one of its pharmaceutically acceptable salts or esters, or of a plant
extract containing it as defined above, which is effective for preventing
or treating allergic manifestations is administered to the said human
being or said animal.
In particular, the abovementioned treatment is applied to the prevention or
symptomatic treatment of allergic or exercise-induced bronchial asthma,
hayfever, spasmodic tracheitis and rhinitis, urticaria and other allergic
eruptions, eczema, red blotches or skin irritations of allergic origin,
pruritus, Quincke's oedema, allergic conjunctivitis and also allergic
reactions of medicinal origin.
According to a variant of embodiment, cucurbitine or one of its
pharmaceutically acceptable salts or esters is administered topically at a
concentration preferably of between 0.001% and 10% by weight.
According to another variant of embodiment, cucurbitine or one of its
pharmaceutically acceptable salts or esters is administered systemically
at a dosage in man of between 0.1 mg/kg/day and 20 mg/kg/day, and
preferably between 1 mg/kg/day and 15 mg/kg/day.
The invention further relates to a process for preparing a cosmetic or
pharmaceutical, in particular dermatological, composition, characterized
in that cucurbitine or one of its cosmetically or pharmaceutically
acceptable salts or esters, or a plant extract containing it as defined
above, is incorporated in a cosmetically or pharmaceutically acceptable
carrier, vehicle or excipient.
According to a variant of embodiment, cucurbitine or one of its
cosmetically or pharmaceutically acceptable salts or esters is
incorporated in a complete cosmetic or pharmaceutical formulation for
decreasing the risk of the latter being allergenic.
According to another variant of embodiment, a composition having
antiallergic activity is prepared. Some variants of preparation also
result from the foregoing description.
According to a particular embodiment of the invention in the context of any
one of the aspects stated above, the abovementioned composition containing
cucurbitine or one of its salts or esters, or the abovementioned plant
extract, contains, in addition, vesicles of the liposome type. According
to a particular variant, the cucurbitine, its salt or ester is at least
partially encapsulated in vesicles of the liposome type. The expression
"vesicle of the liposome type" is understood to mean both hydrated
lamellar lipid phases and lipid vesicles composed of ionic or nonionic
amphiphilic lipids. Also, the expression "to incorporate at least
partially in vesicles of the liposome type" is understood, in the present
description and the claims, to mean that the cucurbitine, its salt or
ester is combined with vesicles of the liposome type irrespective of the
form of this combination. However, a preferred combination lies in
encapsulation of the cucurbitine, its salt or ester in vesicles of the
liposome type. However, it is not necessary for the total amount to be
incorporated or encapsulated in order to obtain the desired antiallergic
effect according to the invention.
It is known that vesicles of the "liposome" type are prepared from lipid
substances. The term "lipid" covers all substances comprising a so-called
fatty carbon chain, generally containing more than 5 carbon atoms, this
substance customarily being designated "lipid".
According to the invention, to form either the lamellar lipid phases or the
vesicles of the liposome type, amphiphilic lipids, that is to say lipids
consisting of molecules possessing a hydrophilic group which can be
equally well ionic or nonionic and a lipophilic group, are used as lipids,
these amphiphilic lipids being capable of forming lamellar lipid phases or
vesicles of the liposome type in the presence of an aqueous phase.
In particular, among these lipids, there may be mentioned: phospholipids,
phosphoaminolipids, glycolipids, polyoxyethylenated fatty alcohols and
optionally polyoxyethylenated esters of a polyol. Such substances consist,
for example, of an optionally hydrogenated egg or soya bean lecithin, a
phosphatidylcholine, a phosphatidylserine, a sphyngomyelin, a cerebroside
or an oxyethylenated polyglycerol stearate.
Incorporation of the compounds used according to the present invention in
hydrated lamellar lipid phases or in liposomes may be carried out
according to known preparation techniques, described, for example, in the
document U.S. Pat. No. 4,508,703, and, where appropriate, in combination
with the document U.S. Pat. No. 4,621,023.
According to a seventh aspect, the present invention also covers a process
for the synthesis of cucurbitine, characterized in that
1-benzyl-3-pyrrolidinone is used as starting material.
According to a particular variant of embodiment of this synthesis process,
1-benzyl-3-pyrrolidinone is treated with an ammoniacal solution of
ammonium chloride and of potassium cyanide to obtain
(.+-.)-3-amino-1-benzyl-3-cyanopyrrolidine. This compound is then
converted by acid or basic hydrolysis to
(.+-.)-3-amino-1-benzyl-3-pyrrolidinecarboxylic acid, and lastly a
reduction with hydrogen, preferably a catalytic hydrogenolysis, is carried
out to obtain (.+-.)-3-amino-3-pyrrolidinecarboxylic acid or
(.+-.)-cucurbitine.
According to a preferred variant, the abovementioned ammoniacal solution is
an aqueous-alcoholic solution, the alcohol advantageously being
isopropanol or methanol.
According to another preferred variant, the abovementioned hydrolysis is
performed using 6N aqueous hydrobromic acid solution.
According to yet another variant, the catalytic hydrogenolysis is performed
in water under hydrogen at atmospheric pressure in the presence of a
catalyst such as palladium on charcoal dispersed in the aqueous reaction
medium.
According to a preferred embodiment of the synthesis process,
1-benzyl-3-pyrrolidinone is allowed to react with ammonium chloride and
potassium cyanide in a 1:4:4 mole ratio at room temperature for at least
48 h.
According to another particular variant of embodiment, the optical isomers
are separated from the racemic mixture according to any separation
technique known to a person skilled in the art, and especially via the
preparation of diastereoisomers.
The process for the synthesis of cucurbitine according to the invention
leads to especially high yields of racemic cucurbitine, on average 2 to 3
times as high as those of the known processes.
Other objects, features and advantages of the present invention will become
clearly apparent in the light of the explanatory description which follows
and which is given with reference to various examples of preparation of
cucurbitine, as well as to various examples reporting the results of
pharmacological tests, as well as various examples of cosmetic or
pharmaceutical formulation. In the examples, the percentages are given by
weight except where otherwise stated.
EXAMPLE 1
Synthesis of Cucurbitine in the Form of a Racemic mixture
The procedure is as follows:
a) Synthesis of (.+-.)-3-amino-1-benzyl-3-cyanopyrrolidine
0.5 g of 1-benzyl-3-pyrrolidinone (2.85 mmol) dissolved in 3 ml of
2-propanol is added to a solution of 0.741 g (11.4 mmol) of potassium
cyanide and 0.615 g (11.4 mmol) of ammonium chloride in 7 ml of 28%
ammonia solution. The mixture remains at room temperature with stirring
for 3 days. The solution is washed with 15 ml of 10% potassium carbonate
solution and extracted with dichloromethane (3xc 15 ml). After drying over
magnesium sulphate and evaporation of the solvents, an oil (0.475 g) is
obtained.
The product is purified on a silica column with solid loading. It is eluted
with a 4:2 ether/petroleum ether mixture.
0.402 g of a beige solid is obtained (yield: 70%), the solid consisting of
3-amino-1-benzyl-3-cyanopyrrolidine having the following NMR spectrum:
.sup.1 HNMR, 300 MHz, CDCl.sub.3 1.8 (broad s, 2H, NH.sub.2); 1.97 (ddd,
1H, J.sub.4,4' =13.4, J.sub.4,5 =8, J.sub.4,5' =5.4); 2.5 (ddd, 1H,
J.sub.4,4' =8, J.sub.4,5' =8, J.sub.4',5' =5.4); 2.64 (d, 1H, J.sub.2,2'
=9.4); 3.04 (d, 1H, J.sub.2',2 =9.4); 3.67 (s, 2H, CH.sub.2 --C.sub.6
H.sub.5); 7.33 (m, 5H, aromatic protons).
b) Synthesis of (.+-.)-3-amino-1-benzyl-3-pyrrolidinecarboxylic acid or
(.+-.)-1-benzylcucurbitine
The hydrolysis of the compound obtained above may be carried out either in
an acid medium or in a basic medium.
Hydrolysis in an acid medium:
0.3 g (1.49 mmol) of 3-amino-1-benzyl-3-cyanopyrrolidine obtained in step
a), dissolved in 5 ml of 48% hydrobromic acid, are brought to
40.degree.-50.degree. C. for 4 h. After evaporation of the acid, the
product is purified on a silica column. The impurities are removed with
CH.sub.2 Cl.sub.2 /MeOH 10% and amino acid is brought off with
MeOH/H.sub.2 O 15%.
After removal of the methanol, the compound is decolorized with animal
charcoal in a minimum amount of aqueous medium in the heated state and
then lyophilized.
A dark yellow solid is obtained (yield: 80%), consisting of
(.+-.)-3-amino-1-benzyl-3-pyrrolidinecarboxylic acid in the form of a
mono- or dihydrobromide having the following NMR spectrum:
.sup.1 HNMR, 300 MHz, D.sub.2 O 2.47-2.69 (m, 1H, H.sub.4); 2.75-2.91 (m,
1H, H.sub.4 '; 3.69-3.94 (m, 3H, H.sub.5, H.sub.5', H.sub.2); 4.12 (d,
J.sub.2,2' =14.2, 1H, H.sub.2'); 4.58 (2d, J=13.8, 2H, CH.sub.2
--CH.sub.5); 7.62 (s, 5H, aromatic protons).
Hydrolysis in a basic medium:
280 mg (1.39 mmol) of 3-amino-1-benzyl-3-cyanopyrrolidine of step a) are
dissolved in 2 ml of ethanol. 5 ml of 10% sodium hydroxide solution are
added and the mixture is then brought to reflux for 5 h. After cooling, it
is acidified with 48% hydrobromic acid.
The product is purified on a silica column: MeOH/H.sub.2 O 10%. The product
is decolorized with animal charcoal and then lyophilized. The presence of
sodium bromide salts causes a yield of greater than 100% to be obtained.
To remove the salts present, 200 mg of pyrrolidine are taken and brought to
pH 8 with 10% sodium hydroxide solution. Duolite Cl.sup.- is loaded with
2N hydrobromic acid solution. The salts are removed with distilled water.
To detach the pyrrolidine, 0.1N hydrobromic acid solution is used.
After lyophilization, 120 mg of
(.+-.)-3-amino-1-benzyl-3-pyrrolidinecarboxylic acid are obtained (yield:
40%).
c) Synthesis of (.+-.)-3-amino-3-pyrrolidinecarboxylic acid or
(.+-.)-cucurbitine
300 mg of (.+-.)-3-amino-1-benzyl-3-pyrrolidinecarboxylic acid hydrobromide
obtained in step b) are dissolved in 10 ml of water. 0.5 mg of charcoal
obtaining 10% of palladium is dispersed, and this suspension is then
placed in a hydrogen atmosphere at atmospheric pressure. Stirring is
maintained for 18 h. After filtration through filter paper and
lyophilization, a yellow solid is obtained (98%), consisting of
(.+-.)-3-amino-3-pyrrolidinecarboxylic acid or (.+-.)-cucurbitine having
the following NMR spectrum:
.sup.1 HNMR, 300 MHz, D.sub.2 O 2.45 (m, 1H, H.sub.4); 2.69 (m, 1H,
H.sub.4'); 3.61 (d, 1H, J.sub.2,2'= 13.4); 3.67-3.78 (m, 2H, H.sub.5,
H.sub.5'); 4 (d, 1H, J.sub.2',2 =13.4).
EXAMPLE 2
Optimization of the Yield of the Synthesis of (.+-.)-cucurbitine
The procedure is as described in Example 1, choosing in step b) hydrolysis
with 6N hydrobromic acid, varying, however, the proportion of the
reactants of step a).
The yields obtained appear in Table 1 below.
TABLE I
______________________________________
Number of
equivalents Experimental
Moles conditions Yield
N.B.P.
NH.sub.4 Cl
KCN of step a) CN COOH
______________________________________
1 1-1 1 6 h room temperature
* <10%
1 1-1 1 4 h 40.degree..INTEGRAL.50.degree.
* <10%
1 4 4 4 h 40.degree..INTEGRAL.50.degree.
* 20%
1 8 8 4 h 40.degree..INTEGRAL.50.degree.
* 20%
1 4 4 48 h room temperature
66% 44%
1 4 4 72 h room temperature
70% 49%
1 4 4 84 h room temperature
70% 56%
______________________________________
N.B.P.: 1benzyl-3-pyrrolidinone
*aminonitrile, not isolated
CN: yield of 3amino-1-benzyl-3-cyanopyrrolidine
COOH--: overall yield of 3amino-3-pyrrolidinecarboxylic acid
The last column of Table I contains the yields of (.+-.)-cucurbitine
relative to the starting reactants.
It is observed that the yields are maximal if the reaction is carried out
at room temperature for a period exceeding 48 h, using proportions of
1:4:4 between the reactants: 1-benzyl-3-pyrrolidone, ammonium chloride and
potassium cyanide.
It was, moreover, observed that hydrolysis by means of hydrobromic acid
also led to better yields than when hydrochloric acid at the same
concentration is used as hydrolysis agent.
EXAMPLE 3
Separation of the Optical Isomers of Cucurbitine
The separation method, which is known per se, is based on the preparation
of diastereoisomeric derivatives by coupling of (.+-.)-cucurbitine with
certain optically active reagents after protection of the acid function by
esterification and of the cyclic amine function. Each form of these
stereoisomers, corresponding to one or other isomer of cucurbitine, will
be isolated by chromatography, in particular by high performance liquid
chromatography or on a silica column. To regenerate thereafter the two
enantiomers of cucurbitine, it suffices to saponify the protecting ester,
then to hydrolyse it in order to liberate the acid function, and to
liberate the amine function, for example by hydrogenolysis as described in
Example 1 in the case where the amine function was protected by
substitution with a benzyl radical.
As reagents for the present method, it is possible to use laevorotatory S
optical active compounds such as:
(1S)-camphanyl chloride,
(S)-(-)-.alpha.-methoxy-.alpha.-(trifluoromethyl)phenylacetic acid,
N-(tert-butoxycarbonyl)-L-phenylalanine.
The present example describes the resolution of the racemate of cucurbitine
by means of coupling with (1S)-camphanyl chloride.
a) Protection of the acid function of (+)-1-benzylcucurbitine: synthesis of
its methyl ester
0.1 g of (.+-.)-1-benzylcucurbitine (0.20 mmol), obtained in step b of
Example 1, is added slowly to an ice-cold solution of thionyl chloride
(0.28 mmol) and methanol (2 ml). The reaction temperature must not rise
above -5.degree. C. The mixture is stirred at 0.degree. C. for 2 h and
then brought back to room temperature for two days.
After evaporation, the product is purified on a silica column with solid
loading. (Eluent CH.sub.2 Cl.sub.2 /MeOH from 10 to 50%).
A yellow product is obtained in a 70% yield, consisting of
(.+-.)-1-benzylcucurbitine methyl ester.
b) Coupling with (1S)-(-)-camphanyl chloride
1 mol of (.+-.)-1-benzylcucurbitine methyl ester, dissolved in 1.5 ml of
methylene chloride, is neutralized with 1 mol of triethylamine. 1.1
equivalents of (1S)-(-)-camphanyl chloride are added. The mixture is left
stirring at room temperature for 16 h.
The product is purified on a silica column with solid loading (eluent:
CH.sub.2 Cl.sub.2 /MeOH 10%).
Separation of the diastereoisomers was performed by HPLC on various
columns, in particular NH.sub.2 -grafted Zorbax.RTM..
EXAMPLE 4
Production of Cucurbitine from Cucurbita pepo Pulp
Fresh Cucurbita pepo fruits are cut in half, and the pips which can be used
for the manufacture of pip extracts are removed. The pulp thereby obtained
is ground and lyophilized. The powder is recovered and defatted with
petroleum ether in the proportion of 1 l to 100 g of powder. The insoluble
matter, which constitutes the desired extract of Cucurbita pepo pulp, is
recovered by filtration. The proportion of cucurbitine in this extract is
assayed by HPLC, and a concentration of 0.03% by weight of cucurbitine is
obtained in the defatted dry extract.
The defatted dry extract is then introduced into water, which is heated to
approximately 50.degree. C. with stirring until dissolution is complete.
An equivalent volume of 95% ethanol is added to precipitate proteinaceous
matter in suspension, which is removed by centrifugation. The clear
supernatant remaining is then acidified to pH 5.0 with 60% perchloric
acid. The solution thus acidified is placed in a refrigerator for at least
2 days to precipitate the cucurbitine perchlorate, which is collected.
Cucurbitine may be obtained from this cucurbitine perchlorate in a
conventional manner for a person skilled in the art, especially by passage
through a cation exchange resin column (Na.sup.+ type) and by evaporation
of the eluate under reduced pressure.
EXAMPLE 5
Preparation of an Extract of Cucurbita pepo Seeds
1.5 kg of previously decorticated Cucurbita pepo seeds are ground. The
powder obtained is subjected three times to extraction with hexane (3
liters, 2 liters and 1.8 liters) to remove fats. The dried cake obtained
is extracted with aqueous hydrochloric acid solution maintained at
approximately pH 4. This extraction is carried out in three stages: two at
room temperature--approximately 22.degree. C.--and the third at 70.degree.
C. At each stage, the time during which the cake-solution is kept in
contact with the hydrochloric acid solution (2 liters on the first
occasion and 2.5 liters on the following two occasions) is 24 h.
After draining, the solid residue is removed and the aqueous phase is
collected, partially evaporated and centrifuged. The centrifugation
pellets are washed with distilled water and then discarded. The washing
liquors are combined with the centrifugation concentrates.
The aqueous fraction is reconcentrated and then treated with an equal
weight of ethanol. A white precipitate forms, which is removed by
centrifugation. The aqueous-alcoholic supernatant, which is neutralized,
for example, with sodium hydroxide, constitutes an extract containing
(-)-cucurbitine, which may be used as it is.
It is also possible to evaporate off the alcohol and then to atomize the
aqueous solution obtained, so as to obtain a powder assaying at 3 to 5% of
(-)-cucurbitine, depending on the batches of seeds used.
EXAMPLE 6
Preparation of a (-)-cucurbitine-rich Extract of Cucurbita pepo Seeds, and
Production of Purified (-)-cucurbitine.
A-preparation of an extract of Cucurbita pepo seeds
The starting material consists either of Cucurbita seeds, decorticated or
otherwise, having a (-)-cucurbitine content of 0.2 to 0.4 per cent by
weight, depending on the batch or origin, or of oil-free cakes of
Cucurbita seeds having a (-)-cucurbitine content of 0.4 to 0.8 per cent by
weight, depending on the batch or origin.
The starting material is preferably ground to approximately 100
micrometers. The ground material is then treated by soaking at room
temperature with acidulated water (sulphuric acid at a concentration of
0.1 per cent by weight) at pH 3.5 approximately, for 2 h in the case of
ground cakes or up to 16 h in the case of seeds. The amount of acidulated
water used is approximately five times the weight of the starting
material. The whole is then brought to boiling at atmospheric pressure for
1 h. After cooling to 80.degree. C., the mixture is filtered and then,
where appropriate, centrifuged. The aqueous phase obtained is then brought
to a temperature of 80.degree. C. to 90.degree. C. and thereafter
microfiltered through a 0.5 micrometer filter. A juice assaying at
approximately 10 g of dry matter per liter is thereby obtained.
This juice then undergoes a thermal preconcentration under a partial vacuum
to a concentration of approximately 250 g/l of dry matter. This
preconcentrate is thereafter placed at 4.degree. C. for 48 h, then
undergoes a further concentration under reduced pressure to 500 g of dry
matter per liter, and is again left standing at 4.degree. C. for 48 h.
These operations are followed by filtration through a filter press. An
extract containing from 40 to 50% of dry matter and assaying at between 1
and 2% of (-)-cucurbitine is thereby obtained. The yields are
approximately 200 l of extract per tonne of starting material.
B-Production of purified (-)-cucurbitine
The abovementioned preconcentrate, assaying at 250 g to 300 g of dry matter
per liter, is neutralized with sodium hydroxide to obtain a pH of 7.5. The
mixture is filtered through a filter press and the filtrate is then passed
through a cation exchange resin (of the Na.sup.+ type). Elution is
performed using ammonia solution. After thermal concentration of the
eluate under a partial vacuum, a syrup is obtained, the dry matter of
which contains approximately 50% by weight of (-)-cucurbitine. If so
desired, this syrup may by lyophilized. For this purpose, it will
advantageously be mixed with a neutral powdery carrier such as talc.
EXAMPLE 7
Demonstration of the Inhibitory Activity of Cucurbitine with Respect to
Histamine Formation
1-A by an Enzymatic Test
This test is based on the inhibitory action of cucurbitine on the enzyme
histidine decarboxylase (HDC), which converts histidine to histamine, in
comparison with that of tritoqualine, which is a known inhibitor of HDC
(see Carpi C., Maggi G. C. Bull. Soc Ital. Sper. 1968, 44 (6 543-4) and
which is used therapeutically as a hypohistaminaemic agent under the name
Hypostamine.RTM..
The inhibitory activity with respect to HDC may be readily assessed by a
colorimetric assay on the basis of the following chemical reaction:
Colorimetric assay
##STR2##
MBTH=3-methyl-2-benzothiazolinonehydrozone
DMA=3-(dimethylamino)benzoic acid
In practice, it is observed that the formation of the blue coloration is
proportional to the concentration of histidine consumed. It has thus been
possible to define initial rates of reaction and to plot so-called
Mickaelis curves as described in Fundamentals of Enzymology, 2nd Ed.
Oxford Univ. Press, 1989.
The initial rate is expressed as a change in absorbance per minute.
The results obtained with various initial concentrations of histidine,
namely 132 nmol, 265 nmol and 530 nmol, respectively without effector,
with tritoqualine as a known HDC inhibitor and with cucurbitine in racemic
form as inhibitor according to the invention, are recorded in Table II
below, and form the subject of the Mickaelis curve which is the subject of
FIG. 1.
TABLE II
______________________________________
Histidine 132 nmol 265 nmol 530 nmol
______________________________________
Without effector
0.062 0.089 0.1132
Tritoqualine 0.021 0.0374 0.0604
(prior art)
Cucurbitine 0.013 0.0242 0.042
(invention)
______________________________________
It emerges clearly from Table II and from the Mickaelis curve which is the
subject of FIG. 1 that cucurbitine is a much more potent HDC inhibitor
than tritoqualine, constituting an altogether surprising result for a
person skilled in the art.
1-B by a RIA assay
The antihistaminic activity of cucurbitine may also be demonstrated by a
radioimmunological assay (Radio Immuno Assay or "RIA") in the following
manner.
This assay takes place by assaying the histamine produced directly under
the action of the enzyme HDC, by the RIA method which is well known to a
person skilled in the art and described, in particular, in the directions
for use of an assay kit called HISTAMINE Radioimmunoassay kit
(Cat..noteq.1302) marketed by the company IMMUNOTECH (Marseilles-France).
The amount of histamine liberated (in nanomoles) for a 16.times.10.sup.-3
molar concentration of histidine in a phosphate buffer at pH 6.3 is
assayed over time, respectively without effector, with tritoqualine as
effector for comparison and with synthetic racemic cucurbitine as
antihistaminic agent according to the invention. Tritoqualine and
cucurbitine are used at a concentration of 2.times.10.sup.-3 molar.
The results obtained, expressed as nanomoles of histidine liberated, are
recorded in Table III below and form the subject of the curve of FIG. 2,
where the number of nanomoles of histamine liberated has been shown as
ordinates and the time expressed in minutes as abscissae. The curve
without effector is plotted as a continuous line, the curve obtained with
tritoqualine is plotted as a chain-dotted line and the curve obtained with
cucurbitine is plotted as a dotted line.
TABLE III
______________________________________
% I* (at
% I* (at
Time 5 min** 10 min** 5 min)
10 min)
______________________________________
Without 32.66 140.79 0 0
effector
Tritoqualine
16.48 49.27 49 65
Cucurbitine
14.85 38.29 55 73
______________________________________
*I = Inhibition
**nanomoles of histamine liberated
It is clearly seen from Table III that the amount of histamine liberated is
markedly lower in the presence of cucurbitine.
The results obtained by the RIA method hence confirm that cucurbitine has a
markedly more potent inhibitory activity with respect to histamine
formation than tritoqualine after a few minutes.
Various examples of formulation of cosmetic or pharmaceutical, in
particular dermatological, compositions according to the invention are as
follows:
EXAMPLE 8
Per tablet for oral administration:
______________________________________
Per tablet for oral administration:
______________________________________
(.+-.)-cucurbitine 100 mg
starch 38 mg
lactose 75 mg
talc 10 mg
other excipients for tablets
250 mg
(including magnesium stearate) qs
______________________________________
Indications: preventive and curative treatment of allergic manifestations,
in particular cutaneous and respiratory manifestations.
Dosage: 1 to 10 tablets per day for adults. Decrease the dosage by 2 for
children up to 15 years of age.
EXAMPLE 9
Powder for Aerosol
Per 100 g in a pressurized can:
______________________________________
Per 100 g in a pressurized can:
______________________________________
(.+-.)-cucurbitine
3 g
mannitol 1 g
propellent gases
96 g
______________________________________
Indications: preventive and curative treatment of all respiratory allergic
manifestations such as bronchial asthma and asthmoid bronchitis.
Dosage: intrabronchial administration, at the rate of 4 to 6 inhalations
per day on average.
______________________________________
Example 10
Demulcent emulsion for sensitive skin
pip extract according to Example 5
10 g
assaying at 3% of (-)-cucurbitine
conventional cosmetic emulsion
100 g
for sensitive skins (fatty alcohols,
mineral oil, isopropyl palmitate,
glycerol, gelling agent, preservatives,
fragrances, water) qs
Example 11
Hypoallergenic make-up foundation
extract of Cucurbita pepo pulp,
10 g
fortified with (-)-cucurbitine,
according to Example 4, having a
content of 1% of (-)-cucurbitine
conventional make-up foundation
100 g
compoisiton (fatty esters, squalane,
soya bean lecithin, volatile silicone,
propylene glycol, xanthan gum,
sunscreen agent, pigments,
preservatives, fragrance, water) qs
______________________________________
EXAMPLE 12
Anti-irritant Mascara
______________________________________
(.+-.)-cucurbitine 1.0 g
ultramarine blue 9.0 g
hexadecyl alcohol 7.4 g
propylene glycol 9.0 g
stearic acid 11.3 g
glycerol monostearate 4.4 g
triethylamine 3.6 g
preservative 0.3 g
water qs 100 g
______________________________________
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